This invention relates to the chemical synthesis of nucleosides, non-nucleosides and derivatives thereof, including nucleoside and non-nucleoside phosphoramidites and succinates.
The following is a brief description of the synthesis of nucleosides. This summary is not meant to be complete but is provided only for understanding the invention that follows. This summary is not an admission that the work described below is prior art to the claimed invention.
Structural modifications of oligonucleotides are becoming increasingly important as their possible clinical applications emerge (Usman et al, 1996, Ed., Springer-Verlag, Vol. 10, 243-264; Agrawal, 1996, Trends Biotech., 14, 376-387; Christoffersen and Marr, 1995, J. Med. Chem., 38, 2023-2037). The efficient synthesis of nucleic acids that are chemically modified to increase nuclease resistance while maintaining potency is of importance to the potential development of new therapeutic agents.
Research into the study of structure-function relationships in ribonucleic acids has in the past, been hindered by limited means of producing such biologically relevant molecules (Cech, 1992, Nucleic Acids Research, 17, 7381-7393; Francklyn and Schimmel, 1989, Nature, 337, 478-481; Cook et al., 1991, Nucleic Acids Research, 19, 1577-1583; Gold, 1988, Annu. Rev. Biochemistry, 57, 199-233). Although enzymatic methods existed, protocols that allowed one to probe structure function relationships were limited. Only uniform post-synthetic chemical modification (Karaoglu and Thurlow, 1991, Nucleic Acids Research, 19, 5293-5300) or site-directed mutagenesis (Johnson and Benkovic, 1990, The Enzymes, Vol. 19, Sigman and Boyer, eds., 159-211) were available In the latter case, researchers were limited to using natural bases. Fortunately, adaptation of the phosphoramidite protocol for RNA synthesis has greatly accelerated our understanding of RNA. Site-specific introduction of modified nucleotides at any position in a given RNA has now become routine. Furthermore, one is not confined to a single modification but can include many variations in each molecule.
While it is seemingly out of proportion that one small structural modification can have such an impact, the presence of a single hydroxyl at the 2xe2x80x2-position of the ribofuranose ring has been the major reason that research in the RNA field has lagged so far behind comparable DNA studies. Progress has been made in improving methods for DNA synthesis that have enabled the production of large amounts of antisense deoxyoligonucleotides for structural and therapeutic applications. Only recently have similar gains been achieved for RNA (Wincott et al., 1995, Nucleic Acids Research, 23, 2677-2684; Sproat et al., 1995, Nucleosides and Nucleotides, 14, 255-273; Vargeese et al., 1998, Nucleic Acids Research, 26, 1046-1050).
The chasm between DNA and RNA synthesis is due to the difficulty of identifying orthogonal protecting groups for the 5xe2x80x2- and 2xe2x80x2-hydroxyls. Historically, two standard approaches have been taken by scientists attempting to solve the RNA synthesis problem, The first approach involves developing a method that seeks to adapt to state-of the-art DNA synthesis, while the second approach involves designing a method specifically suited for RNA. Although adaptation of the DNA process provides a more universal procedure in which non-RNA phosphoramidites can easily be incorporated into RNA oligomers, the advantage to the latter approach is that one can develop a process that is optimal for RNA synthesis and as a result, better yields can be realized. However, in both cases similar issues exist, including, for example, the identification of protecting groups that are both compatible with synthesis conditions and capable of being removed at the appropriate juncture. This problem does not refer only to the 2xe2x80x2- and 5xe2x80x2-OH groups, but includes the base and phosphate protecting groups as well. Consequently, the accompanying deprotection steps, in addition to the choice of ancillary agents, are critical. Another shared obstacle is the need for efficient synthesis of the monomer building blocks.
The most common paradigm has been to apply DNA synthesis methods to RNA. Consequently, it is critical to identify a 2xe2x80x2-hydroxyl protecting group that is compatible with DNA protecting groups yet can easily be removed once the oligomer is synthesized. Due to constraints placed by the existing amide protecting groups on the bases and the 5xe2x80x2-O-dimethoxytrityl (DMT) group (or in some cases the 9-(phenyl)xanthen-9-yl (Px) group), the 2xe2x80x2-blocking group must be stable to both acid and base. In addition, the 2xe2x80x2blocking group must also be inert to the oxidizing and capping reagents. Although the most widely used 2xe2x80x2-hydroxyl protecting group is tert-butyldimethylsilyl (TBDMS) ether, many others have been explored. These alternative 2xe2x80x2-protecting groups include acetal groups, such as the tetrahydropyranyl (THP), methoxytetrahydropyranyl (mthp), 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), 1-(2-chloroethoxy)ethyl, 2-hydroxyisophthalate formaldehyde acetal, and 1-{4-[2-(4-nitrophenyl)ethoxycarbonyloxy]-3-fluorobenzyloxy}ethyl groups. In addition, photolabile groups, such as the o-nitrobenzyl, o-nitrobenzyloxymethyl, and p-nitrobenzyloxymethyl groups have been used. Other groups include the 1,1-dianisyl-2,2,2-trichloroethyl group, the p-nitrophenylethyl sulfonyl group, and the 2xe2x80x2-O-triisopropylsilyl-oxy-methyl group. Additional 2xe2x80x2-protecting groups that have been studied are reviewed in Gait et al., 1991; Oligonucleotide Synthesis, In Oligonucleotides and Analogues, A Practical Approach (F. Eckstein, ed.), 25-48, and Beaucage and Iyer, 1992, Tetrahedron, 48, 2223-2311.
By far the most popular 2xe2x80x2-protecting group is the tert-butyldimethylsilyl group, developed principally by Ogilvie and co-workers (Usman et al., 1987, J.A.C.S., 109, 7845-7854). Recent advances in silyl chemistry in both the synthesis (Wincott et al., 1995, Nucleic Acids Research, 23, 2677-2684, Sproat et al., 1995, Nucleosides and Nucleotides, 14, 255-273, Vargeese et al., 1998, Nucleic Acids Research, 26, 1046-1050) and deprotection (Wincott et al., supra; Sproat et al., supra) arenas have made it""s use an even more viable approach to the production of oligoribonucleotides.
The introduction of the tert-butyldimethylsilyl group at the 2xe2x80x2-position of a ribonucleotide is usually effected by the reaction of a 5xe2x80x2-O-dimethoxytrityl-nucleoside with tert-butyldimethylsilyl chloride in the presence of either silver nitrate or imidazole. The resulting mixture of 2xe2x80x2-O-tert-butyldimethylsilyl, 3xe2x80x2-O-tert-butyldimethylsilyl and bis-substituted (3xe2x80x2,2xe2x80x2-di-O-tert-butyldimethylsilyl) products must be purified to isolate the desired 2xe2x80x2-O-tert-butyldimethylsilyl derivative, usually by column chromatography. Treatment of the isolated 3xe2x80x2-O-tert-butyldimethylsilyl derivative by equilibration in triethylamine/methanol or pyridine/water can effect migration of the silyl ether, resulting in the capability of isolating additional 2xe2x80x2-O-tert-butyldimethylsilyl product. Multiple re-equilibrations can be utilized to obtain smaller and smaller quantities of the desired 2xe2x80x2-O-tert-butyldimethylsilyl product, however, this process is time-consuming and requires a separate purification step after each equilibration. Therefore, even though formation of the 2xe2x80x2-O-tert-butyldimethylsilyl isomer is sometimes kinetically favored, the resulting isolated yield of the desired 2xe2x80x2-O-tert-butyldimethylsilyl isomer is generally diminished due to formation of the competing 3xe2x80x2-O-tert-butyldimethylsilyl and bis-substituted isomers. Accordingly, there exists a need for a general method for nucleoside phosphoramidite synthesis useful in the selective introduction of silyl protection at the 2xe2x80x2-hydroxyl of a nucleoside.
The utilization of 2xe2x80x2-deoxy-2xe2x80x2-amino nucleotides has resulted in the in vitro selection of novel enzymatic nucleic acid molecules (Beaudry et al., 2000, Chemistry and Biology, 7, 323-334). As such, there exists a need for methods suitable for the efficient synthesis of 2xe2x80x2-deoxy-2xe2x80x2-amino containing oligonucleotides. Beigelman et al., 1995, Nucleic Acids Res., 23, 4434-4442, have previously shown that the use of the phthaloyl protecting group for the 2xe2x80x2-amino function of a 2xe2x80x2-deoxy-2xe2x80x2-amino ribonucleotide phosphoramidite during oligonucleotide synthesis is preferable to trifluoroacetyl or Fmoc protecting groups. Beigelman et al., supra, also describe the synthesis of 2xe2x80x2-N-phthaloyluridine phosphoramidite starting from 2xe2x80x2-aminouridine using Nefkins"" method (Nefkins et al., 1960, Recl. Trav. Chim. Pays-Bas., 79, 688-698). This procedure requires 2xe2x80x2-deoxy-2xe2x80x2-amino-nucleosides as starting materials.
The first preparation of 2xe2x80x2-aminouridine was described by Verheyden et al., 1971, J. Org. Chem., 36, 250-254. This procedure utilizes lithium azide in the opening of 2,2xe2x80x2-O-anhydrouridine in 50% yield followed by catalytic reduction to the corresponding amine. Several reports elaborating this approach with minor modifications have since been published. An approach utilizing intramolecular cyclization of the 3xe2x80x2-O-trichloroacetimidate of 2,2xe2x80x2-O-anhydrouridine, followed by acid hydrolysis has been published as an alternative to the use of azide ion (McGee et al., 1996, J. Org. Chem., 61, 781-785). Methods for the synthesis of the 2xe2x80x2-aminopurine nucleosides use the same general strategy of introducing a 2xe2x80x2-azido group with subsequent reduction to the amine. Alternatively, 2xe2x80x2-azidopurine nucleosides have been prepared by glycosylation with 2xe2x80x2-azido-2xe2x80x2-deoxy ribose derivatives (Hobbs and Eckstein, 1977, J. Org., Chem., 42, 714-719), transglycosylation with 2xe2x80x2-amino-2xe2x80x2-deoxyuridine, (Imazawa and Eckstein, 1979, J. Org. Chem., 44, 2039-2041), opening of 8,2-cyclopurine nucleosides with azide ion, (Ikehara et al., 1977, Chem. Pharm. Bull., 25, 754-760; Ikehara and Maruyama, 1978, Chem. Pharm. Bull., 26, 240-244), and by displacement of the corresponding 2xe2x80x2-arabino triflates with azide ion (Robins et al., 1992, Nucleosides and Nucleotides, 11, 821-834).
Other publications have described the preparation of nucleoside derivatives, including, for example, Karpeisky et al., International PCT Publication No. WO 98/28317, which describes the synthesis of 2xe2x80x2-O-amino nucleosides, Beigelman et al., U.S. Pat. No. 5,962,675, which describe the synthesis of 2xe2x80x2-O-methyl nucleosides, Furusawa, Japanese patent No. 6067492, which describes the synthesis of nucleoside cyclic silicon derivatives, Furusawa, Japanese patent No. 10226697, which describes the synthesis of 2xe2x80x2-O-silyl nucleosides, Usman et al, U.S. Pat. No. 5,631,360, which describes N-phthaloyl protected 2xe2x80x2-amino nucleoside phosphoramidites, Usman et al., U.S. Pat. No. 5,891,683, describe non-nucleoside containing enzymatic nucleic acid molecules, and Matulic-Adamic et al., U.S. Pat. No. 5,998,203, describe enzymatic nucleic acid molecules containing 5xe2x80x2 and/or 3xe2x80x2-cap structures.
The invention provides a universal method for the synthesis of 2xe2x80x2-deoxy-2xe2x80x2-amino purine and pyrimidine nucleosides and C-nucleosides that employs fewer synthetic steps, avoids the use of azides, and which concomitantly introduces N-phthaloyl protection of the 2xe2x80x2-amine (see FIG. 1).
In one embodiment, the present invention provides a method for the preparation of 2xe2x80x2-deoxy-2xe2x80x2-amino and 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleosides. The method can be scaled up to kilogram or greater quantities. The method comprises the use of phthalimide and/or a substituted phthalimide as a nucleophile in the displacement of a leaving group present at the 2xe2x80x2-position of a 1-xcex2-D-arabinofuranosyl nucleoside, to generate a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside. Subsequent cleavage of the phthaloyl protection with a suitable base results in the formation of a 2xe2x80x2-deoxy-2xe2x80x2-amino nucleoside.
The present invention provides a method for synthesizing a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside, comprising: (a) introducing a leaving group at the 2xe2x80x2-position of a 1-xcex2-D-arabinofuranosyl nucleoside; an (b) displacing said leaving group from step (a) with a phthalimide or substituted phthalimide nucleophile to yield 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside.
In another embodiment, the invention provides a method for synthesizing a 2xe2x80x2-deoxy-2xe2x80x2-amino nucleoside, comprising the steps of: (a) introducing a leaving group at the 2xe2x80x2-position of a 1-xcex2-D-arabinofuranosyl nucleoside; (b) displacing said leaving group from step (a) with a phthalimide or substituted phthalimide nucleophile to yield a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside; and (c) deprotecting said 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside to yield said 2xe2x80x2-deoxy-2xe2x80x2-amino nucleoside.
In another embodiment, the present invention provides a method for the preparation of 2xe2x80x2-deoxy-2xe2x80x2-amino and 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleosides. The method can be scaled up to kilogram or greater quantities. The method comprises the use of phthalimide and/or a substituted phthalimide as a nucleophile in the displacement of a leaving group present at the 2xe2x80x2-position of a 1-xcex2-D-arabinofuranosyl C-nucleoside, to generate a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleoside. Subsequent cleavage of the phthaloyl protection with a suitable base results in the formation of a 2xe2x80x2-deoxy-2xe2x80x2-amino C-nucleoside.
In another embodiment, the invention provides a method for synthesizing a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside, comprising the step of contacting a 2xe2x80x2-trifluoromethanesulfonyl-1-xcex2-D-arabinofuranosyl nucleoside with a phthalimide or substituted phthalimide nucleophile under conditions suitable for formation of said 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside.
In another embodiment, the invention provides a method for synthesizing a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleoside, comprising the step of contacting a 2xe2x80x2-trifluoromethanesulfonyl-1-xcex2-D-arabinofuranosyl C-nucleoside with a phthalimide or substituted phthalimide nucleophile under conditions suitable for formation of said 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleoside.
In another embodiment, the invention provides a method for the synthesis of a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside, comprising the step of contacting a 2xe2x80x2-methanesulfonyl-1-xcex2-D-arabinofuranosyl nucleoside with a phthalimide or substituted phthalimide nucleophile under conditions suitable for formation of said 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl nucleoside.
In another embodiment, the invention provides a method for the synthesis of a 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleoside, comprising the step of contacting a 2xe2x80x2-methanesulfonyl-1-xcex2-D-arabinofuranosyl C-nucleoside with a phthalimide or substituted phthalimide nucleophile under conditions suitable for formation of said 2xe2x80x2-deoxy-2xe2x80x2-N-phthaloyl C-nucleoside.
In another aspect, the invention also provides a method for the synthesis of nucleic acid base protected 2xe2x80x2-O-silyl nucleoside phosphoramidites and 2xe2x80x2-O-silyl C-nucleosides (FIG. 2) that avoids formation of the competing 3xe2x80x2-O-silyl nucleoside isomer, thereby improving overall synthetic yield while avoiding the need for separation of 2xe2x80x2-O-silyl nucleoside and 3xe2x80x2-O-silyl nucleoside isomers. The method described herein avoids the practice of re-equilibration of the 3xe2x80x2-O-silyl nucleoside isomer to generate additional 2xe2x80x2-O-silyl nucleoside. Additionally, the present method avoids the need for transient protection of the furanosyl hydroxyls as a separate step in the protection of the nucleic acid base.
The present invention also provides a method for the preparation of 2xe2x80x2-O-silyl-nucleosides and 2xe2x80x2-O-silylnucleoside phosphoramidites. The method can be scaled up to kilogram or greater quantities. The method comprises the steps of (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to a nucleoside; (2) introducing a 2xe2x80x2-O-silyl protecting group to the product of step (1); (3) introducing nucleic acid base protection where necessary to the product of step (2); (4) selectively desilylating the product of step (3); (5) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (4), and (6) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (5) with a phosphitylating reagent to yield a 2xe2x80x2-O-silyl-nucleoside phosphoramidite.
In another embodiment, the invention provides a method for the synthesis of 2xe2x80x2-O-silyl-nucleosides and 2xe2x80x2-O-silyl-nucleoside phosphoramidites comprising the steps of (1) introducing nucleic acid base protection where necessary to a nucleoside; (2) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to the product of step (1); (3) introducing a 2xe2x80x2-O-silyl protecting group to the product of step (2); (4) selectively desilylating the product of step (3); (5) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (4); and (6) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (5) with a phosphitylating reagent to yield a 2xe2x80x2-O-silyl-nucleoside phosphoramidite.
In another embodiment, the method for synthesis of 2xe2x80x2-O-silyl-nucleosides and 2xe2x80x2-O-silyl-nucleoside phosphoramidites is used for the synthesis of 2xe2x80x2-O-silyl-D-ribofuranosyl nucleosides, 2xe2x80x2-O-silyl-D-ribofuranosyl nucleoside phosphoramidites, 2xe2x80x2-O-silyl-L-ribofuranosyl nucleosides, 2xe2x80x2-O-silyl-L-ribofuranosyl nucleoside phosphoramidites, 2xe2x80x2-O-silyl-D-arabinofuranosyl nucleosides, 2xe2x80x2-O-silyl-D-arabinofuranosyl nucleoside phosphoramidites and both 2xe2x80x2-O-silyl-L-arabinofuranose nucleosides and 2xe2x80x2-O-silyl-L-arabinofuranose nucleoside phosphoramidites.
The present invention also provides a method for the preparation of 2xe2x80x2-O-silyl-C-nucleosides and 2xe2x80x2-O-silyl-C-nucleoside phosphoramidites. The method can be scaled up to kilogram or greater quantities. The method includes the steps of (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to a C-nucleoside; (2) introducing a 2xe2x80x2-O-silyl protecting group to the product from step (1); (3) introducing nucleic acid base protection where necessary to the product of step (2); (4) selectively desilylating the product of step (3); (5) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (4); and (6) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (5) with a phosphitylating reagent.
In another embodiment, the invention provides a method for synthesizing 2xe2x80x2-O-silyl-C-nucleosides and 2xe2x80x2-O-silyl-C-nucleoside phosphoramidites comprising the steps of (1) introducing nucleic acid base protection where necessary to a C-nucleoside; (2) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to the product of step (1); (3) introducing a 2xe2x80x2-O-silyl protecting group to the product from step (2); (4) selectively desilylating the product of step (3); (5) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (4); and (6) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (5) with a phosphitylating reagent.
In another embodiment, the method for synthesis of 2xe2x80x2-O-silyl-C-nucleosides and 2xe2x80x2-O-silyl-C-nucleoside phosphoramidites is used for the synthesis of 2xe2x80x2-O-silyl-D-ribofuranosyl C-nucleosides and 2xe2x80x2-O-silyl-D-ribofuranosyl C-nucleoside phosphoramidites, 2xe2x80x2-O-silyl-L-ribofuranosyl C-nucleosides and 2xe2x80x2-O-silyl-L-ribofuranosyl C-nucleoside phosphoramidites, 2xe2x80x2-O-silyl-D-arabinofuranosyl C-nucleosides and 2xe2x80x2-O-silyl-D-arabinofuranosyl C-nucleoside phosphoramidites and both 2xe2x80x2-O-silyl-L-arabinofuranose C-nucleosides and 2xe2x80x2-O-silyl-L-arabinofuranose C-nucleoside phosphoramidites.
In yet another aspect of the invention, a method for the preparation of 2xe2x80x2-O-methyl guanosine nucleosides and 2xe2x80x2-O-methyl guanosine nucleoside phosphoramidites is provided. The 2xe2x80x2-O-methyl guanosine nucleosides and 2xe2x80x2-O-methyl guanosine nucleoside phosphoramidites are synthesized from a 2,6-diaminopurine nucleoside by selective methylation of the 2,6-diaminopurine nucleoside followed by selective deamination of the 2,6-diaminopurine nucleoside to afford a 2xe2x80x2-O-methyl guanosine nucleoside.
The present invention provides a practical method for the preparation of 2xe2x80x2-O-methyl guanosine nucleosides and 2xe2x80x2-O-methyl guanosine nucleoside phosphoramidites. The method can be scaled up to kilogram or greater quantities. The method includes the steps of (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to a 2,6-diamino-9-(xcex2-ribofuranosyl)purine with a disilylalkyl bis(trifluoromethanesulfonate) to form a 2,6-diamino-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (2) methylation of the product of step (1) under conditions suitable for the isolation of a 2,6-diamino-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-xcex2-ribofuranosyl]purine; (3) introducing acyl protection at the N2 and N6 positions of the product from step (2) under conditions suitable for the isolation of N2-N6-2,6-diamino-diacyl-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (4) selectively deacylating position N6 of the product of step (3), under conditions suitable for the isolation of 2,6-diamino-N2-acyl-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (5) chemically deaminating the N6-amine and desilylating the product of step (4), under conditions suitable for the isolation of N2-acyl-2xe2x80x2-O-methyl guanosine; (6) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (5), under conditions suitable for obtaining a N2-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-methyl guanosine; and (7) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (6) with a phosphitylating reagent under conditions suitable for isolating a N2-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-methyl guanosine 3xe2x80x2-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite).
In another embodiment, the present invention provides a method for the chemical synthesis of a 2xe2x80x2-O-methyl guanosine nucleoside comprising the steps of: (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to a 2,6-diamino-9-(xcex2-ribofuranosyl)purine with a disilylalkyl bis(trifluoromethanesulfonate) to form a 2,6-diamino-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-xcex2-ribofuranosyl]purine; (2) methylation of the product of step (1) under conditions suitable for the isolation of a 2,6-diamino-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (3) acylation of the N2 and N6 positions of the product from step (2) under conditions suitable for the isolation of a 2,6-diamino-N2-N6-diacyl-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (4) selectively deacylating position N6 of the product of step (3), under conditions suitable for the isolation of a 2,6-diamino-N2-acyl-9-[5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-methyl-xcex2-ribofuranosyl]purine; (5) deaminating the N6-amine and desilylating the product of step (4) under conditions suitable for the isolation of a N2-acyl-2xe2x80x2-O-methyl guanosine; and (6) deprotection of the N2-amine from the product of step (e) under conditions suitable for the isolation of said 2xe2x80x2-O-methyl guanosine nucleoside.
In yet another aspect of the invention, a method for the preparation of 2xe2x80x2-O-alkyl adenosine nucleosides and 2xe2x80x2-O-alkyl adenosine nucleoside phosphoramidites is provided. The 2xe2x80x2-O-alkyl adenosine nucleosides and 2xe2x80x2-O-alkyl adenosine nucleoside phosphoramidites are synthesized from a adenosine by selective alkylation of the 2xe2x80x2-hydroxyl of 5xe2x80x2,3xe2x80x2-silanediyl protected adenosine nucleoside followed by selective deprotection of the 5xe2x80x2,3xe2x80x2-silanediyl to afford a 2xe2x80x2-O-alkyl adenosine nucleoside. Protection of the N6 amine of adenosine if desired can take place after alkylation and before deprotection of the 5xe2x80x2,3xe2x80x2-silanediyl to afford a N6-acyl-2xe2x80x2-O-alkyl adenosine. Acid labile protecting groups and phosphorous containing groups compatible with oligonucleotide synthesis can be introduced as is known in the art.
In one embodiment, the 2xe2x80x2-O-alkyl adenosine nucleosides and 2xe2x80x2-O-alkyl adenosine nucleoside phosphoramidites are synthesized from a inosine by introducing an imidazole or triazole moiety at the O6 position of a 5xe2x80x2,3xe2x80x2-silanediyl protected inosine nucleoside as, followed by selective alkylation of the 2xe2x80x2-hydroxyl of the 5xe2x80x2,3xe2x80x2-silanediyl protected adenosine N6-imidazole nucleoside followed by N6 amination and deprotection of the 5xe2x80x2,3xe2x80x2-silanediyl and to afford a 2xe2x80x2-O-alkyl adenosine nucleoside. Alternately, the 5xe2x80x2,3xe2x80x2-silanediyl protected 2xe2x80x2-O-alkyl adenosine N6-imidazole nucleoside is desilyated to a 2xe2x80x2-O-alkyl adenosine N6-imidazole nucleoside which is aminated with ammonia to provide 2xe2x80x2-O-alkyl adenosine. Acid labile protecting groups and phosphorous containing groups compatible with oligonucleotide synthesis can be introduced as is known in the art.
The present invention provides a practical method for the preparation of 2xe2x80x2-O-alkyl adenosine nucleosides and 2xe2x80x2-O-alkyl adenosine nucleoside phosphoramidites. The method can be scaled up to kilogram or greater quantities. The method includes the steps of (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to adenosine with a disilylalkyl bis(trifluoromethanesulfonate) to form a 5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-adenosine; (2) alkylation of the product of step (1) under conditions suitable for the isolation of a 5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-alkyl adenosine; (3) introducing acyl protection at the N6 position of the product from step (2) under conditions suitable for the isolation of N6-acyl-5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-alkyl adenosine; (4) desilylating the product of step (3), under conditions suitable for the isolation of N2-acyl-2xe2x80x2-O-alkyl adenosine; (5) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (4), under conditions suitable for obtaining a N6-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-alkyl adenosine; and (6) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (5) with a phosphitylating reagent under conditions suitable for isolating a N6-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-alkyl adenosine 3xe2x80x2-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite).
In another embodiment, the present invention provides a method for the chemical synthesis of a 2xe2x80x2-O-alkyl adenosine nucleoside comprising the steps of: (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to adenosine with a disilylalkyl bis(trifluoromethanesulfonate) to form a 5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl) adenosine; (2) alkylation of the product of step (1) under conditions suitable for the isolation of a 5xe2x80x2,3xe2x80x2-O-(di-alkylsilanediyl)-2xe2x80x2-O-alkyl adenosine; (3) desilylating the product of step (2), under conditions suitable for the isolation of N6-acyl-2xe2x80x2-O-alkyl adenosine.
The present invention provides a practical method for the preparation of 2xe2x80x2-O-alkyl adenosine nucleosides and 2xe2x80x2-O-alkyl adenosine nucleoside phosphoramidites. The method can be scaled up to kilogram or greater quantities. The method includes the steps of (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to inosine to form a 5xe2x80x2,3xe2x80x2-protected-inosine; (2) introducing a N6 imidazole moiety to the product of step (1) under conditions suitable for the isolation of a 5xe2x80x2,3-protected-N6-imidazole adenosine; (3) alkylation of the product of step (2) under conditions suitable for the isolation of a 5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl-N6-imidazole adenosine; (4) introducing acyl protection at the N6 position of the product from step (3) under conditions suitable for the isolation of N6-acyl-5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl adenosine; (5) desilylating the product of step (4), under conditions suitable for the isolation of N6-acyl-2xe2x80x2-O-alkyl adenosine; (6) introducing a 5xe2x80x2-hydroxyl protecting group to the product of step (5), under conditions suitable for obtaining a N6-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-alkyl adenosine; and (7) introducing a phosphoramidite moiety at the 3xe2x80x2-position of the product of step (6) with a phosphitylating reagent under conditions suitable for isolating a N6-acyl-5xe2x80x2-O-dimethoxytrityl-2xe2x80x2-O-alkyl adenosine 3xe2x80x2-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite).
In another embodiment, the present invention provides a method for the chemical synthesis of a 2xe2x80x2-O-alkyl adenosine nucleoside comprising the steps of: (1) introducing a 5xe2x80x2,3xe2x80x2-cyclic silyl protecting group to inosine to form a 5xe2x80x2,3xe2x80x2-protected inosine; (2) introducing a N6 imidazole moiety to the product of step (1) under conditions suitable for the isolation of a N6-imidazole-5xe2x80x2,3xe2x80x2-protected adenosine; (3) alkylation of the product of step (2) under conditions suitable for the isolation of a N6-imidazole-5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl adenosine; (4) aminating the N6 position of the product from step (3) under conditions suitable for the isolation of a N6-acyl-5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl adenosine or 5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl adenosine; (5) desilyl product of step (4), under conditions suitable for the isolation of N6-acyl-2xe2x80x2-O-alkyl adenosine or 2xe2x80x2-O-alkyl adenosine.
In another embodiment, amination of the N6-imidazole-5xe2x80x2,3xe2x80x2-protected-2xe2x80x2-O-alkyl adenosine utilizes an acylamide, for example benzamide, to introduce exocyclic amine protection, either before or after desilylation.
The present invention also provides a practical method for the synthesis of 1,4-anhydro-2-deoxy-D-erythro-pentitol derivatives, including 1,4-anhydro-2-deoxy-D-erythro-pentitol succinates and phosphoramidites. The method includes the steps of (1) depyrimidination of a 5xe2x80x2-O-protected thymidine derivative under conditions suitable for the isolation of a 5-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol, (2) introduction of an acid-labile protecting group at the C3 hydroxyl of the 5-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol under conditions suitable for the isolation of a 5-O-protected-3-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol, (3) selective 5-O-deprotection of the product of step (2) under conditions suitable for the isolation of a 3-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol, and (4) introducing a chemical moiety comprising a succinate moiety or a phosphoramidite moiety at position 5 of the product of step (3) under conditions suitable for the isolation of a 5-O-succinyl-3-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol or a 3-O-protected-1,4-anhydro-2-deoxy-D-erythro-pentitol-5-O-phosphoramidite.